There has been an explosion of biological information in the last few years resulting from the use of high throughput experimental techniques. These techniques range from genome sequencing, microarray analysis, yeast 2-hybrid protein-protein interaction assays, to RNAi screens and the use of automated image analysis to study cell biological processes.
Following the successful implementation of each ‘-omic’ technique, opportunities and bottlenecks are created at the interface between one set of techniques and the next. One key bottleneck is that of biochemistry.
Molecular machines, formed from complexes of proteins, are the building blocks underlying most cellular processes. Techniques such as the yeast 2-hybrid technique have been used to identify binary interactions between pairs of proteins on a genome-wide scale, while complementary analyses using complex purification and mass spectrometry are starting to identify the combinations of components that comprise each molecular machine. However, the detailed study of biochemical interactions requires the identification of affinity binding and rate constants, together with techniques for studying how these properties are physiologically regulated.
Standard ‘test tube’ biochemical techniques, including calorimetry and fluorescence anisotropy, require the time-consuming production and purification of microgram to milligram quantities of soluble proteins and are therefore unlikely to be scaled-up for high throughput applications. A potentially more promising technique, Surface Plasmon Resonance (Biacore®) requires somewhat less protein and can tolerate the presence of impurities (in some formats), but requires the careful timed flow of assay protein, followed by wash solutions over an immobilized binding partner and is therefore unlikely to be easily adapted for high throughput analyses. Typical protein requirements for Surface Plasmon Resonance and calorimetry are described below:
Isothermal Titration Calorimetry:
Measures:Kd, Stoichiometry (n), ΔG, ΔNRequires:1 ml of 10 μM solution; 20 nMoles; 1 mg of 50 KDa protein.Surface Plasmon Resonance (Biacore®)
Measures:Kon, Koff, KdRequires:2 mls of 100 nM solution; 200 pMoles; 10 μg of 50KDa protein*.*Calculation based on typical series of experiments required to establish a binding affinity in the ~10 nM range.
The high levels of proteins that are required in these assays result from the requirement to ‘saturate’ binding ligand concentrations 4-10× higher than the dissociation constant. This imposes the typical requirements shown in Table 1.
TABLE 1Kd4 × Kdμg/ml for 50 KDa protein1nM4nM0.210nM40nM2100nM400nM201μM4μM200
These levels of protein require time consuming and relatively expensive conventional protein synthesis and purification systems.
In vitro translation ‘pull-down’ experiments can be used to identify binding interactions between small amounts of radiolabelled protein produced in a translation extract (100-150 ng produced). However, like the yeast 2-hybrid technique, they suffer from the disadvantage that they are non-quantitative, they screen for picomolar to low nanomolar ranges of affinities and fail to screen for lower affinity interactions.
There is a requirement for new high-throughput biochemical techniques that are quantitative, sensitive, may use small volumes of analyte, may require a low number of moles of analyte, may achieve high (up to mid-micromolar typically about 1-5 pM to about 10-20 μM) concentrations and may be adaptable for massively parallel analyses.